This invention relates to gene delivery. More particularly, this invention relates to compositions of matter, methods of use thereof, and methods of making thereof for delivering genes.
Gene therapy provides significantly important opportunities for treating various kinds of life-threatening and gene-related disease by producing biologically active agents or stopping abnormal functions of the cells, such as genetic failure or uncontrollable proliferation of cells. Actual application of genes to human therapy is limited by several problems, including their instability in body fluids, non-specificity to the desired cells, degradation by nucleases, and low transfection efficiency. Gene delivery systems have been investigated in attempts to enhance gene expression and reduce cytotoxicity. S.-O. Han et al., Development of Biomaterials for Gene Therapy, 2 Mol. Ther. 302-317 (2000).
Among the various gene delivery systems, viral vectors, M. A. Rosenfield et al., Adenovirus-mediated Transfer of a Recombinant A1-antitrypsine Gene to the Lung Epithelium In Vivo, 252 Science 431-434 (1991); H. M. Temin, Safety Considerations in Somatic Gene Therapy of Human Disease with Retrovirus Vectors, 1 Hum. Gene Ther. 111-123 (1990), liposomal carriers, X. Gao & L. Huang, Cationic Liposome-mediated Gene Delivery, 2 Gene Ther. 710-722 (1995); A. R. Thierry et al., Systemic Gene Delivery: Biodistribution and Long-term Expression of a Transgene in Mice, 92 Proc. Nat'l Acad. Sci. USA 9742-9746 (1995); J. H. Senior et al., Interaction of Positively-charged Liposomes with Blood: Implications for Their Application In Vivo, 1070 Biochim. Biophys. Acta 173-179 (1991), and cationic polymers, Y.-B. Lim et al., Biodegradable Polyester, Poly [α-(4-amino-butyl)-L-glycolic acid], as a Non-toxic Gene Carrier, 17 Pharm. Res. 811-816 (2000); P. Lemieux et al., Block and Graft Copolymers and NanoGel Copolymer Networks for DNA Delivery into Cell, 8 J. Drug. Target. 91-105 (2000), S.-O. Han et al., Water Soluble Lipopolymer for Gene Delivery, 12 Bioconjug. Chem. 337-345 (2001), have been widely investigated in gene therapy areas. Although retroviruses, adenoviruses, and adeno-associated viruses have shown higher transfection efficiency in vitro, the application of viral vectors to the human body is also limited by safety problems such as the immune response against transfection systems, oncogenic effects, and the potential ability of endogenous virus recombination. These problems have stimulated the development of non-viral gene delivery. As non-viral vectors, liposomes and cationic polymers have been extensively investigated for a decade due to the advantages of safety and relatively low cost. Although higher transfection efficiency has been reported by liposomal gene carriers in vitro, A. R. Thierry et al., supra, some liposomal gene carriers are unstable in aqueous solution and aggregate in blood. J. H. Senior et al., supra. Cationic polymers including poly(L-lysine) (“PLL”) and polyethyleneimine (“PEI”) were able to condense plasmid DNA and protect it from enzymatic degradation, which resulted in enhancement of transfection efficiency. However, drawback, such as biocompatibility in the body, still remain before such polymers can be used for gene delivery. To overcome the biocompatibility problem, non-toxic biodegradable polymeric gene carriers have been developed as promising gene delivery materials. Y.-B. Lim et al., supra. However, the biodistribution of the polymer/pDNA complexes following the injection of complexes into the body is still unknown. For the enhanced delivery of genes to specific cells, polymeric gene carriers have been modified with specific cell targeting moieties such as galactose, M. Nishikawa et al., Hepatocyte-targeted In Vivo Gene Expression by Intravenous Injection of Plasmid DNA Complexed with Synthetic Multi-functional Gene Delivery System, 7 Gene Ther. 548-555 (2000), transferrin, E. Wagner et al., Influenza Virus Hemaglutinin HA-2 N-terminal Fusogenic Peptides Augment Gene Transfer by Transferrin-polylysine-DNA complexes: Toward a Synthetic Virus-like Gene-transfer Vehicle, 89 Proc. Nat'l Acad. Sci. USA 7934-7938 (1992), and antibody, W. Suh et al., Anti-JL1 Antibody Conjugated Poly(L-lysine) for Targeted Gene Delivery to Leukemia T Cells, 72 J. Control. Release 171-178 (2001).
Recently, a series of methoxy poly(ethylene glycol)-grafted-poly(L-lysine (PEG-g-PLL) gene carriers was synthesized for reducing cytotoxicity, increasing solubility in aqueous solution, and enhancing the transfection efficiency resulting from long-term expression compared to PLL in a human carcinoma cell line. Y. H. Choi et al., Polyethylene Glycol-grafted Poly-L-lysine as Polymeric Gene Carrier, 54 J. Control. Release 39-48 (1998). A lactose group was also coupled to the end of PEG-g-PLL for specific targeting to hepatoma cells. Y. H. Choi et al., Lactose-poly(ethylene glycol)-grafted Poly-L-lysine as Hepatoma Cell-targeted Gene Carrier, 9 Bioconjug. Chem. 708-718 (1998); Y. H. Choi et al., Characterization of a Targeted Gene Carrier, Lactose-Polyethylene Glycol-grafted Poly-L-lysine, and its Complex with Plasmid DNA, 10 Hum. Gene Ther. 2657-2665 (1999). Transfection efficiency of such Lac-PEG-g-PLL/pDNA complexes was increased several-fold higher than that of PLL/DNA complexes in Hep G2 cells. A7R5 and NIH 3T3 cell lines do not have lactose receptors on their surfaces; consequently, the transfection efficiency of Lac-PEG-g-PLL/pDNA complexes was much lower than in the Hep G2 cells.
It was well known that low-density lipoprotein (LDL) can be taken up by different types of cells (vascular endothelial cells, vascular smooth muscle cells, hepatocytes, and macrophages) via receptor-mediated endocytosis. In previous reports, J. S. Kim et al., In Vitro Gene Expression on Smooth Muscle Cells Using a Terplex Delivery System, 47 J. Control. Release 51-59 (1997); J. S. Kim et al., Terplex DNA Delivery System as a Gene Carrier, 15 Pharm. Res. 116-121 (1998), a terplex-DNA gene delivery system comprising plasmid DNA, low density lipoprotein (LDL), and hydrophobized poly(L-lysine) (H-PLL) enhanced gene transfer via the LDL receptor-mediated endocytosis pathway. The transfection efficiency of the terplex-DNA system was 2-5 times higher than that of Lipofectin™/pDNA in A7R5 murine smooth muscle cells. Lipofectin™ reagent is a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water. This system also showed significantly higher transfection efficiencies in vitro in artery wall cells, L. Yu et al., Terplex DNA Gene Carrier System Targeting Artery Wall Cells, 72 J. Control. Release 179-189 (2001), and in vivo in myocardium cells, D. G. Affleck et al., Augmentation of Myocardia Transfection Using Terplex DNA: a Novel Gene Delivery System, 8 Gene Ther. 349-353 (2001).
Gene delivery systems containing a specific cell-targeting moiety have the advantage in the efficient delivery to the desired cells or organs. Although PLL has been described as an efficient gene carrier, U. K. Laemmli, Characterization of DNA Condensates Induced by Poly(ethylene oxide) and Polylysine, 72 Proc. Nat'l Acad. Sci. USA 4288-4299 (1975), as an alternative to liposomes or viral vectors, PLL/pDNA complexes displayed some limitations such as the precipitation of PLL/pDNA complexes in high concentration and low biocompatibility in the human body. Y. H. Choi et al., 54 J. Control. Release 39-48 (1998), investigated PEGylated-PLL/pDNA complexes to overcome these limitations of PLL by conjugation of PEG to PLL. Although PEGylated-PLL was shown to be a biocompatible material in tissues, efficient transfection to specific cells still remained a problem to overcome.
In view of the foregoing, it will be appreciated that providing a composition for matter for specific gene delivery to artery wall cells would be a significant advancement in the art.